Destructible surfactants and uses thereof

ABSTRACT

The present invention provides methods for enhancing chemical reactions of molecules, e.g., biomolecules, with destructible surfactants. The chemical reactions may involve and/or be associate with analysis, e.g., solubilizing, separating, purifying and/or characterizing the molecules. In one aspect, the anionic surfactants of the present invention may be selectively broken up at relatively low pH. The resulting breakdown products of the surfactants may be removed from the molecule/sample with relative ease. The invention has applicability in a variety of analytical techniques.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/385,021, filed on May 31, 2002 (Attorney Docket No. WCZ-032-1).This application is related to U.S. Patent Application No. 60/134,113,filed on May 14, 1999 (Attorney Docket No. WCZ-004-1), and published PCTInternational application No. WO 00/70334, published Nov. 23, 2000(Attorney Docket No. WAA-213 PCT; application No. PCT/US00/13028, filedon May 12, 2000). The entire contents of the aforementioned applicationsare hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Surfactants are used in a variety of applications. For example,surfactants are used commercially for cleaning manufactured items,removing paints, chemical processing, for use in emulsionpolymerization, solubilizing drugs, purifying proteins, and variousbioanalytical applications.

In addition, surfactants have been employed in chemical alterationreactions, e.g., reduction or alkylation, involving biomolecules, suchas proteins, for solubilization, or the surfactants are present in thereaction as an artifact of the process of preparation, e.g.,electrophoresis. Reactions, e.g., reduction and alkylation, of largeproteins are important steps for in-solution digestion because of theirability to increase the number of peptide fragments. Organic salts, suchas urea and detergents, e.g., sodium dodecylsulfate (SDS), are commonlyused to solubilize protein mixtures before reduction and alkylation.However, urea and SDS inhibit trypsin activity, and therefore theirconcentrations have to be diluted prior to in-solution trypsindigestions. Additionally, it is known that if digestion, in which ureahas been utilized as a solubilizing agent, is allowed to proceed for toolong, the urea will act upon and modify the protein, making analysis ofpeptide fragments more difficult and inaccurate.

Furthermore, SDS and urea are also known to suppress the MS signal ifthey are not removed. The additional sample preparation steps that arerequired for current methodologies decrease the reliability andsensitivity of the analysis, especially for low abundance proteins.

Another particular bioanalytical application that uses surfactants issodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Inthe past three decades, SDS-PAGE has been widely used as a simple andrelatively rapid tool for analysis and purification of large moleculessuch as proteins (U.K. Laemrnli, Nature 227, 680-685, 1970). Sodiumdodecylsulfate (SDS) is an anionic surfactant that denatures proteins byforming a stable complex. Upon denaturation, SDS binds to most proteinsand peptides in a constant weight ratio of about 1.4:1. As a result, theSDS-protein complexes have almost identical charge densities andtherefore migrate in a polyacrylamide gel according to molecular weight.If the gel is of the correct porosity, a plot of log M_(w) vs. relativemobility, R_(f), results in a linear relationship. The band intensityafter staining is a rough indicator of the amount present in the sample.When coupled with another electrophoretic technique,isoelectricfocusing, SDS-PAGE can separate complex mixtures into severalhundred discrete components.

The ability to estimate the size and amount of a protein has led tovarious applications of SDS-PAGE. However, there are some drawbacks tothe technology. For example, it is very difficult to use massspectrometry to monitor and analyze samples from SDS-PAGE separationsbecause SDS interferes with the sensitivity of mass spectrometrydetection. Furthermore, it is very difficult to separate SDS fromSDS/protein complex since SDS is a surfactant that forms emulsions.

Protein digestion to produce protein fragments is an important aspect ofprotein characterization. Currently, the rate-limiting step in massspectrometric analysis of protein fragments is the extended timerequired for digestion, e.g., typically 12 hours or more as in the caseof trypsin digestion of proteins. Furthermore, the large amounts oftrypsin required in current protocols can result in increased backgroundnoise due to, trypsin autolysis. In addition, the current approaches totrypsin digestion result in mass spectrometric identification of only alimited number of the peptide fragments, e.g., about 60%.

It is also known in the art that trypsin digestion can be acceleratedby: (1) performing the digestion at elevated temperatures (Anal. Chem.2001, 73, 2558-2564); (2) in the presence of certain organic solvents(Anal. Chem. 2001, 73, 2682-2685); or (3) using immobilized trypsin.However, these methods often result in miscleavages, or are difficult toreproduce. Therefore, when it is desirable to generate a reproduciblepeptide map, the preferred methodology is overnight incubation, oftendone at low temperature. Nevertheless, a method for enhancing (e.g.,acceleration with high reproducibility and low miscleavage) thedigestion of proteins is desired.

SUMMARY OF THE INVENTION

The present invention provides methods for enhancing chemical reactionsof molecules, e.g., biomolecules, with destructible surfactants. Thechemical reactions may involve and/or be associated with analysis, e.g.,solubilizing, separating, purifying, detecting and/or characterizing themolecules. For example, in one embodiment, a compound of the invention,e.g., sodium 4-[(2-methyl-2 undecyl-1dioxolan-4-yl)methyl]-1-propanesulfonate (ALS), may be useful as anadditive, e.g., denaturant, for separation of intact proteins andpeptides in various techniques of HPLC (i.e., such that the separationusing HPLC may be considered the chemical reaction). In one aspect, theanionic surfactants of the present invention may be selectively brokenup at relatively low pH. The resulting breakdown products of thesurfactants may be removed from the sample with relative ease.

The invention has applicability in a variety of techniques that benefitfrom the initial presence and ultimate removal of a surfactant.Moreover, the surfactants of the invention allow for more rapid,reproducible, relatively low temperature, e.g., room temperature,protease, e.g., trypsin, digestion of a biomolecule, e.g., protein,which requires less protease due to enhanced efficiency of the reactionwithout concomitant increase in miscleavages. In fact, the digestions ofthe present invention are more complete than those of currentmethodology and therefore afford an increased number of correctlycleaved peptide fragments.

The elimination of excess trypsin, surfactants, and organic solventsfrom the protease digestions eliminates mass spectrometric suppressionand reduces background noise which would otherwise be due to trypsinautolysis, therefore improving analysis of low abundance biomolecules.The reduction of time required for digestion further lends itself torapid on-line, automated digestion and analysis.

In addition, the elimination of sample preparation steps required withcurrent methodology increases the reliability and sensitivity of theanalysis, especially for low abundance proteins. In certain embodiments,the surfactants of the present invention will not act upon and will notmodify the protein, reducing the difficulty of analysis of peptidefragments, as compared, e.g., with the use of urea as a surfactant.

Accordingly, one embodiment of the invention provides a method forenhancing a chemical reaction of a molecule comprising contacting themolecule with a surfactant represented by formula I:

in which

p is 0, 1 or 2;

R is alkyl;

R₁ and R₂ are each, independently, hydrogen or methyl; and

R₃ is selected from −OSO₃, —R₄OSO₃ ⁻, —R₄OR₅SO₃ ⁻, and —OR₅SO₃ ⁻,wherein R₄ and R₅ are each, independently, lower alkyl;

to thereby enhance the chemical reaction of the molecule.

In another aspect, the invention pertains a method for analysis of abiomolecule comprising: enhancing a chemical reaction of the biomoleculeby contacting a sample containing the biomolecule with a surfactantrepresented by Formula I, and analyzing the sample to thereby analyzethe biomolecule.

Another aspect of the invention provides a kit for enhancing a chemicalreaction of a molecule comprising a surfactant represented by formula I,and instructions for use.

In yet another aspect, the invention provides a method of capturing alipophilic compound comprising contacting a lipophilic compound with asurfactant represented by formula (I), and degrading the surfactant toproduce a hydrophobic compound, to thereby capture the lipophiliccompound.

In an additional aspect, the invention provides a method for enhancingsurface desorption ionization analysis of a molecule comprising:contacting the molecule with a surfactant represented by formula (I), tothereby analyze the sample by surface desorption ionization.

In yet another aspect, the invention provides a method for enhancingchemical digestion of a biomolecule comprising: contacting the moleculewith a digestive enzyme and a surfactant represented by formula I, tothereby enhance the chemical digestion of the molecule.

Another aspect of the invention provides a kit for enhancing chemicaldigestion of a biomolecule comprising a surfactant represented byformula I, and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the analysis to determine the trypsin activity, indicatingthat SDS inhibits trypsin activity at each the percentages examined.

FIGS. 2A-2B show the LC/MS TIC of tryptic digested myoglobin solubilizedwith (A) 0.1% ALS or (B) 50 mM NH₄HCO₃ (no ALS).

FIGS. 3A-3B show the MALDI-TOF mass spectrometry analysis of the trypticdigestion of myoglobin after 5 minutes solubilized with 0.1% ALS or 50mM NH₄HCO₃ (no ALS).

FIGS. 4A-4C show the mass spectra of the myoglobin comparative digestion(in the presence and absence of ALS), using ° (4A) Asp-N, (4B) Lys-C,and (4C) Glu-C performed in Example 4.

FIGS. 5A-5B show the mass spectrometry analysis of the disulfide bondreduction of lysozyme C in the presence and absence of 025% ALS.

FIGS. 6A-6C show electrospray mass spectra of myoglobin under thevarious treatment conditions described in Example 7. FIG. 6A is thespectrum of 5 μM myoglobin in 50/50 20 mM ammonium acetate/water with 1%acetic acid. FIG. 6B is the spectrum of 5 μM myoglobin in 50/50 20 mMammonium acetate/water with 1% acetic acid and 0.1% SDS. FIG. 6C is thespectrum of 5 μM myoglobin in 50/50 20 mM ammonium acetate/water with 1%acetic acid and 0.1% ALS that had reacted in 10% acetic acid for 16hours.

DETAILED DESCRIPTION OF THE INVENTION Overview of the Invention

The invention provides methods for enhancing chemical reactions ofmolecules, e.g., biomolecules, with destructible surfactants. Thechemical reactions may involve and/or be associated with analysis, e.g.,solubilizing, separating, purifying, detecting and/or characterizing themolecules. For example, in one embodiment, a compound of the invention,e.g., ALS, may be useful as an additive, e.g., denaturant, forseparation of intact proteins and peptides in various techniques of HPLC(i.e., such that the separation using HPLC may be considered thechemical reaction). In particular, the invention includes anionicsurfactants with binding and electrophoretic properties similar to SDS.However, the surfactants of the present invention, unlike SDS, include adioxolane or dioxane functional group that enable degradation of thesurfactant under an acidic environment. The resulting degradant productscan be removed from the sample more readily than the originalsurfactant. In addition, mass spectrometric sensitivity of the moleculesis significantly and surprisingly greater in the presence of thesurfactants of the invention than in the presence of SDS at similarconcentrations, even in the presence of these degradant products. Theinvention has applicability in a variety of techniques that benefit fromthe initial presence and ultimate removal of a surfactant.

Moreover, the surfactants of the invention allow for more rapid,reproducible, relatively low temperature, e.g., room temperature,protease, e.g., trypsin, digestion of a biomolecule, e.g., protein,which requires less protease due to enhanced efficiency of the reactionwithout concomitant increase in miscleavages. In fact, the digestions ofthe present invention are more complete than those of currentmethodology and therefore afford an increased number of correctlycleaved peptide fragments.

The elimination of excess trypsin, surfactants, and organic solventsfrom the protease digestions eliminates mass spectrometric suppressionand reduces background noise which would otherwise be due to trypsinautolysis, therefore improving analysis of low abundance biomolecules.The reduction of time required for digestion further lends itself torapid on-line, automated digestion and analysis.

In addition, the elimination of sample preparation steps required withcurrent methodology increases the reliability and sensitivity of theanalysis, especially for low abundance proteins. In certain embodiments,the surfactants of the present invention will not act upon and will notmodify the protein, reducing the difficulty of analysis of peptidefragments, as compared with the use, e.g., of urea as a surfactant.

Definitions

So that the invention may be more readily understood, the definitions ofa several terms used throughout the specification and claims arepresented here.

The language “sample/surfactant complex” is intended to include acomplex formed by a surfactant of the present invention and a componentof the sample.

The term “molecule” as used herein is intended to include any chemicalentity that may be used within the methods of the present invention.This term is intended to include small molecules and macromolecules,either of which may be classified as a biomolecule.

The term “biomolecule” includes chemical entities that are obtained frombiological sources. Exemplary biomolecules include, but are not limitedto, substances, such as biopolymers, e.g., proteins, e.g., proteolyticproteins or lipophilic proteins, such as receptors and othermembrane-bound proteins, and peptides.

The language “biological sample” refers to any solution or extractcontaining a molecule or mixture of molecules that comprises at leastone biomolecule that is subjected to analysis that originated from abiological source. Biological samples are intended to include crude orpurified, e.g., isolated or commercially obtained, samples. Particularexamples include, but are not limited to, inclusion bodies, biologicalfluids, biological tissues, biological matrices, embedded tissuesamples, and cell culture supernatants.

The language “biological fluid” is intended to include biologicallyproduced fluids such as blood, blood plasma, urine, spinal fluid,mucosal tissue secretions, tears, interstitial fluid, synovial fluid,semen, and breast milk.

The term “lipophilic protein” refers to proteins or peptides that arerelatively hydrophobic. Particular examples include, without limitation,protein from myelin or central nervous system tissue and membrane-boundproteins such as receptors.

The term “receptor” is recognized in the art and refers generally tomembrane-bound molecules, preferably proteins, which bind a ligand andtransmit a signal into the cell. Such receptors usually have anextracellular domain, a trams-membrane domain, and an intracellulardomain.

The term “inclusion body” is recognized in the art and refers to anintracellular structure, preferably one containing an expressed protein.

The language “enhancing surface desorption ionization” is intended toinclude the act of improving the quality of analytical results obtainedfrom surface desorption ionization techniques. The improvement includes,without limitation, enhancement through increased solubility of ananalyte of interest or improved desorption ionization as a result ofdenaturation.

The language “enhancing a chemical reaction” is intended to include theact of influencing a chemical reaction to produce a favorable chemicalproperty.

The language “favorable chemical property” is intended to includeproperties of a chemical reaction of the invention that are advantageousover known methodology. Examples of a favorable chemical propertyinclude, but are not limited to, more complete reaction, e.g., bettercleavage of peptidic fragments resulting from chemical digestion,increased efficiency, i.e., less protease per total protein ratiorequired for digestion, increased yield, increased rate, e.g.,accelerating chemical digestion, e.g., from hours to minutes, e.g.,about 20 hours to about 5 minutes for trypsin digestion, and increasedutility. Examples of favorable chemical properties that relate tochemical reactions that involve analysis may include, withoutlimitation, improved separation, improved purification, increasedsolubilization, increased detection, and improved characterization ofmolecules.

The language “increased utility” is intended to include the use of achemical reaction process on compounds not typically used, or previouslydemonstrated by alternative methods to be unreactive. For example, theuse of the surfactants of the present invention affords the ability tofacilitate digestion, e.g., tryptic digestion, of proteins that aredifficult or impossible to digest by other methods, or facilitatecorrect digestion of proteins that may have occurred by other knownmethods, although with numerous miscleavages.

The terms “denature”, “denaturing” or “denaturation” are usedinterchangeably and intended to include the modification of themolecular structure of a biomolecule, such as a protein or DNA, by thesurfactants of the invention, heat, acid, alkali, or ultravioletradiation, so as to destroy or diminish some of the original properties,e.g., three-dimensional conformation, and especially the specificbiological activity.

The language “chemical reaction” is intended to include a chemicaltransformation or change as the result of an interaction of chemicalentities. In particular embodiments of the invention, the chemicalreaction is chemical digestion or chemical alteration. The chemicalreactions may involve and/or be associated with analysis, e.g.,solubilizing, separating, purifying, detecting and/or characterizing themolecules. For example, in one embodiment, a compound of the invention,e.g., sodium 4-[(2-methyl-2undecyl-1,3-dioxolan-4-yl)methyl]-1-propanesulfonate (ALS), may beuseful as an additive, e.g., denaturant, for separation of intactproteins and peptides in various techniques of HPLC (i.e., such that theseparation using HPLC may be considered the chemical reaction).

The language “chemical digestion” is intended to include a process ofbreaking down a molecule, e.g., a biomolecule, e.g., a protein, intosimpler chemical compounds, e.g., fragments. In one embodiment, thechemical digestion is performed by an enzyme, e.g., a protease, reactionwith CNBr, or reaction with hydroxylamine. Proteases of the presentinvention include both specific, e.g., trypsin, and nonspecific, e.g.,pepsin, or papain, proteases. In certain embodiments, the chemicaldigestion results in breaking of amide bonds. In specific embodiments ofthe invention, the chemical digestion results in breaking designatedamide bonds.

The language “chemical alteration” is intended to include any chemicalreaction of a molecule that is not a chemical digestion. In certainembodiments, the chemical alteration produces a chemically orphysically, e.g., solubilization, altered molecule. In certain otherembodiments, the chemical alteration does not produce a chemically orphysically altered molecule, i.e., catalysis has occurred. In particularembodiments the chemical alteration is alkylation, reduction, e.g.,using dithiothreitol (DTT), or a combination thereof.

The language “solution for degrading the surfactant” refers to anyrelatively low pH solution. Preferably, the pH of the solution isbetween about 0 and about 5, more preferably between about 1 and about3. In general, the lower the pH of the solution for degrading thesurfactant, the less time required to degrade the surfactant. Inaddition, the compound used to make the solution for degrading thesurfactant is not particularly limited: any compound that provides arelatively low pH solution suitable for degrading the surfactants of thepresent invention without damaging the sample is sufficient. Thus, forexample, hydrochloric acid, acetic acid, formic acid, or trifluoroaceticacid (TFA) may be used as the solution for degrading the surfactant. Inparticular embodiments, TFA may be used to degrade the surfactant. Inother particular embodiments, acetic or formic acid may be used as thesolution for degrading the surfactant. In certain embodiments, theremoval of one of the two components of the degraded surfactant may beaccomplished by removing the oily layer formed after degradation.

The term “electrophoresis” refers to any of the various methods ofanalyzing molecules by their rate of movement in an electric field, i.e.based on the charge to mass ratio of the molecules. Examples include,but are not limited to, gel electrophoresis, polyacrylamide gelelectrophoresis, including the tube, slab gel and capillary formats ofpolyacrylamide gel electrophoresis, free zone electrophoresis andcapillary electrophoresis.

The terms “analysis” or “analyzing” are used interchangeably and referto any of the various methods of separating, detecting, isolating,purifying, solubilizing, and/or characterizing molecules, such as, e.g.,intact proteins, peptides, and fragments thereof. Examples include, butare not limited to, solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry, e.g., Matrix AssistedLaser Desorption Ionization-Mass Spec (MALDI-MS) or ElectrosprayIonization (ESI), liquid chromatography, e.g., high performance, e.g.,reverse phase, normal phase, or size exclusion, ion-pair liquidchromatography, liquid-liquid extraction, e.g., accelerated fluidextraction, supercritical fluid extraction, microwave-assistedextraction, membrane extraction, soxhlet extraction, precipitation,clarification, electrochemical detection, staining, elemental analysis,Edmund degradation, nuclear magnetic resonance, infrared analysis, flowinjection analysis, capillary electrochromatography, ultravioletdetection, and combinations thereof.

In certain embodiments, the surfactant of the invention is degradedprior to analysis. In alternative embodiments, the surfactant of theinvention, e.g., ALS, is not degraded prior to analysis. In particular,a surfactant of the invention, such as ALS does not need to be degradedprior to analysis by HPLC or MS (i.e., analysis may be performed priorto degradation). For example, in one embodiment, on-column degradation(e.g., allowing the surfactant to remain at elevated temperatures on thecolumn for longer periods of time, e.g., 20 minutes at 60° C.) may beused to degrade the surfactant during the analysis. Alternatively, thesurfactant of the invention may not require degradation during analysis.

The language “compatible with” is intended to mean that the surfactantsof the invention and/or the degradation products thereof, do notinterfere with and/or enhance the analysis and/or a chemical reaction ofa molecule.

The term “clarification” refers to any process by which insolubleparticulate matter is separated from the liquid phase.

The term “mass spectrometric detection” refers to any of the variousmethods of mass spectroscopy. Examples include, but are not limited to,electrospray ionization (“ESI”), surface desorption ionizationtechniques, and atmospheric pressure chemical ionization (APCI).

The language “surface desorption ionization” is intended to include massspectrometry, such as MALDI-MS, desorption ionization on silicon (DIOS),thermal desorption mass spectrometry, or surface enhanced laserdesorption ionization (SELDI) where desorption ionization isaccomplished on a surface, with or without a matrix assistance.

The language “hydrocarbon” includes substituted or unsubstituted alkyl,alkenyl, alkynyl, or aryl moieties.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₂₀ for straight chain, C₃-C₂₀ for branched chain),and more preferably 20 or fewer. Likewise, preferred cycloalkyls havefrom 3-8 carbon atoms in their ring structure, and more preferably have5 or 6 carbons in the ring structure.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,diallylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chaps of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl(alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenylgroups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. Theterm alkenyl further includes alkenyl groups which include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkenyl group has 6 or fewer carbon atoms in its backbone(e.g., C₁-C₂₀ for straight chain, C₃-C₂₀ for branched chain). Likewise,cycloalkenyl groups may have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₁-C₂₀for straight chain, C₃-C₂₀ for branched chain).

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbon atoms. “Lower alkenyl” and “lower alkynyl” have chainlengths of, for example, 2 to 6 carbon atoms, more preferably 3 or 4carbon atoms.

The term “acyl” includes compounds and moieties that contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkynyl groups, halogens,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups.

Examples of substituted alkoxy groups include halogenated alkoxy groups.The alkoxy groups can be substituted with groups such as alkenyl,alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,diallylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “amine” or“amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups that includearyl or heteroaryl moieties bound to an amino group that is bound to thecarbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “carbonyl” or “carboxy” includes compounds and moieties thatcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties that contain a carbonyl include aldehydes, ketones,carboxylic acids, amides, esters, anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietiesthat contain a carbon connected with a double bond to a sulfur atom.

The term “ester” includes compounds and moieties that contain a carbonor a heteroatom bound to an oxygen atom that is bonded to the carbon ofa carbonyl group.

The term “ester” includes alkoxycarboxy groups such as methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.The alkyl, alkenyl, or alkynyl groups are as defined above.

The term “ether” includes compounds or moieties that contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom that is covalentlybonded to another alkyl group.

The term “thioether” includes compounds and moieties that contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom that is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomthat is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated,” perfluorinated, generally refers to a moiety,e.g., perfluorocarbons, wherein all hydrogens are replaced by halogenatoms, e.g., fluorine.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, alkylaminoacarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

Compounds of the Invention

The destructible surfactants of the invention may be prepared as shownin Scheme 1 set forth in Example 1 below. These surfactants havefunctionality similar to sodium dodecylsulfate (SDS) but, unlike SDS,they may be hydrolyzed in aqueous acid solution under mild condition togive two nonsurfactant products: an ionic, water-soluble compound and aneutral, water-insoluble, hydrophobic compound.

In one embodiment, the anionic surfactants of the invention have thestructure of the general formula (Formula I):

in whichp is 0, 1 or 2;R is alkyl;R₁ and R₂ are each, independently, hydrogen or methyl; andR₃ is selected from −OSO₃ ⁻, —R₄OSO₃ ⁻, —R₄OR₅SO₃ ⁻, and —OR₅SO₃ ⁻;wherein R₄ and R₅ are each, independently, lower alkyl.

In certain embodiments, the surfactants have the structure of Formula I,with the provisos that when p is 0 and R₁ is methyl, R₃ is not—CH₂O(CH₂)₄SO₃ ⁻ or, when p is 1 and R₁ is hydrogen and R₂ is methyl, R₃is not —CH₂OSO₃.

In particular embodiments, p is 0 or 1. In other particular embodiments,R is an alkyl having from six to twenty carbon atoms, more specificallyfrom eight to eighteen carbon atoms, and more specifically from ten tosixteen carbon atoms. In certain embodiments, R₃ is —R₄OSO₃ ⁻, —R₄OR₅SO₃⁻, or —OR₅SO₃, and most preferably R₃ is —CH₂O(CH₂)₃SO3₃ ⁻ or—CH₂—O(CH₂)₄SO3₃ ⁻. In certain embodiments, R₄ and R₅ are each,independently, an alkyl group having from one to eight carbons, morespecifically from two to six carbon atoms, and more specifically, threeor four carbon atoms.

In another embodiment, the anionic surfactants of the invention have thestructure of general formula (Formula II):

in which

R₆ is alkyl;

R₇ is selected from —OSO₃ ⁻, —R₄OSO₃, —R₄OR₅SO₃ ⁻, and —OR₅SO₃ ⁻,wherein R₄ and R₅ are each, independently, lower alkyl.

In certain embodiments, the surfactants of the present invention havethe structure of Formula II, with the proviso that when R₆ is —C₉H₁₉,—C₁₁H₂₃, or —C₁₃H₂₇, R₇ is not —CH₂—O—(CH₂)₄SO₃ ⁻.

In particular embodiments, the surfactant of the invention has thefollowing chemical structure:

As indicated in more detail in the Examples, the methods of synthesis ofthe present invention produce isomers. Although the methods of usingsurfactants of the invention do not require separation of these isomers,such separation may be accomplished, if desired, by methods known in theart. For example, preparative high performance liquid chromatographymethods may be used for isomer purification.

Methods of the Invention

The surfactants of the present invention may be used in applicationsthat benefit from the initial presence and ultimate removal of asurfactant. In particular, the present invention is useful for methodsfor enhancing chemical reactions of molecules, e.g., biomolecules, withdestructible surfactants. The chemical reactions may involve and/or beassociated with analysis, e.g., solubilizing, separating, purifying,detecting and/or characterizing the molecules.

Accordingly, one embodiment of the invention provides a method forenhancing a chemical reaction of a molecule comprising contacting themolecule with a surfactant of the present invention to thereby enhancethe chemical reaction of the molecule.

In another embodiment, the invention is a kit for enhancing a chemicalreaction of a molecule comprising a surfactant of the present inventionand instructions for use.

Yet another embodiment is directed to a method for analysis of abiomolecule comprising enhancing a chemical reaction of the biomoleculeby contacting a sample containing the biomolecule with a surfactant ofthe present invention and analyzing the sample to thereby analyze thebiomolecule.

In certain embodiments of the invention, the molecule is a biomolecule.In addition, the biomolecule may be contained in a biological sample,for example, inclusion bodies, biological fluids, biological tissues,biological matrices, embedded tissue samples, or cell culturesupernatants. Examples of biomolecules include peptides and proteins,e.g., lipophilic proteins, receptors, membrane-bound proteins, orproteolytic proteins.

In certain embodiments, the method for enhancing a chemical reaction mayadditionally comprise an analysis of the molecule, e.g., following thechemical reaction. Moreover, analysis of the molecule, e.g.,biomolecule, following the chemical reaction, may include, but is notlimited to, solid phase extraction; solid phase micro extraction;electrophoresis, capillary electrophoresis or gel electrophoresis, e.g.,polyacrylamide gel electrophoresis, e.g., tube, slab gel or capillaryformats, free zone electrophoresis; mass spectrometry, e.g., MALDI-MS orESI; liquid chromatography, e.g., high performance, e.g., reverse phase,normal phase, or size exclusion, ion-pair liquid chromatography;liquid-liquid extraction, e.g., accelerated fluid extraction;supercritical fluid extraction; microwave-assisted extraction; membraneextraction; soxhlet extraction; precipitation; electrochemicaldetection; staining; elemental analysis; Edmund degradation; ultravioletdetection; clarification; nuclear magnetic resonance; infrared analysis;flow injection analysis; capillary electrochromatography; andcombinations thereof. In specific embodiments, the analysis furthercomprises separating the resulting biomolecule fragments.

In certain embodiments, the mass spectrometry analysis is surfacedesorption ionization (SDI) mass spectrometry. Although removal of thedegraded surfactant is easily accomplished, it is not required forSDI-MS. In fact, in SDI-MS analysis, degradation of the surfactant isnot required prior to analysis. In this regard, mass spectrometricanalysis has shown that a 250 fmol ACTH sample spotted with 0.25%surfactant of the invention onto a MAI surface gave virtually the samesignal intensity and quality as when the sample was applied withoutsurfactant. Furthermore, although degradation is required forelectrospray ionization mass spectrometry, it is not necessary to removethe degraded surfactant prior to analysis.

The chemical reaction involving the molecule may include chemicaldigestion, chemical alteration, or a combination thereof. Moreover, thechemical reaction with the molecule, e.g., biomolecule, or analysis ofthe molecule may involve denaturation, solubilization, or a combinationthereof. Additionally, in certain embodiments the enhancement of thechemical reaction facilitates on-line automation, separation, massspectrometric analysis, or a combination thereof. Furthermore, thechemical reaction may also be performed under microscale conditions,e.g., reactions may performed within the spot used for SDI analysis.After the chemical reaction proceeds, the surfactant of the presentinvention involved in enhancing the chemical reaction can be degraded,e.g., by contact with an acidic solution. For example, sodium4-[(2-methyl-2 undecyl-1,3-dioxolan-4-yl)methyl]-1-propanesulfonate(ALS), the synthesis of which is shown in Example 1, while stable at pH7-8 (suitable pH for trypsin digestion), may be degraded at low pH:

In particular embodiments, the chemical reaction using surfactants ofthe present invention is chemical digestion. In one embodiment, thechemical digestion occurs by contacting a molecule, e.g., a biomolecule,with a protease. Exemplary proteases include, but are not limited tospecific proteases such as Trypsin, Chymotrypsin Lys-C, Glu-C (V8protease), AspN, Arg-C, S. Aureus, and Clostripain, and non-specificproteases, such as, Pepsin, and Papain. In certain embodiments of theinvention the protease is immobilized, e.g., immobilized enzymaticreactor. Alternatively, digestion may be accomplished by reaction withCNBr or reaction with hydroxylamine. Additionally, in certainembodiments of the invention, the digestion can occur in anelectrophoretic gel in the presence or absence of one or moresurfactants that are different from the surfactants of the invention,e.g., SDS.

Solubilization of insoluble samples by surfactants of the presentinvention allows digestion of samples that are typically insoluble andtherefore difficult to digest by known methodology. In addition, thesurfactants of the present invention enable the use of lower amounts oftrypsin to digest a protein. For example, the ratio of trypsin to totalprotein is typically 1:50 to 1:20. However, in the presence of thesurfactants of the present invention, the ratio of trypsin to totalprotein can be 1:100 or lower. Moreover, the use of the surfactants ofthe invention, for example, enables rapid tryptic digestion without aconcomitant increase in miscleavages, as observed for knownmethodologies, e.g., addition of organic solvent or heat.

Alternatively, the chemical reaction using surfactants of the presentinvention is chemical alteration. Exemplary chemical alterationsinclude, but are not limited to, alkylation, reduction, e.g., using DTT,and a combination thereof. In fact, the surfactants of the invention,unlike the organic salts, such as urea and detergents, e.g., SDS, usedfor solubilization prior to reduction and alkylation do not inhibit theactivity of proteases, e.g., trypsin. Therefore, in certain embodimentsthe resulting reaction mixtures require no additional sample preparationsteps prior to in-solution digestion.

Additionally, it is known that if digestion, in which urea has beenutilized as a solubilizing agent, is allowed to proceed for too long,the urea will act upon and modify the protein, making analysis ofpeptide fragments more difficult. In this regard, the surfactants of theinvention will not modify the protein, regardless of the length of timeallowed for digestion.

In certain embodiments, the enhancement of the chemical reactioncomprises a favorable chemical property. Exemplary favorable chemicalproperties include, without limitation, a more complete reaction,increased efficiency, increased yield, increased rate, and increasedutility.

The sensitivity of mass spectrometric detection of proteins in thepresence of degraded ALS is much greater than in the presence of SDS.The anionic surfactants of the present invention provide surprisingadvantages over SDS when analyzing molecules. For example, in FIG. 6B,which depicts the mass spectrum of myoglobin treated with SDS, nosignals due to myoglobin are observed. In contrast, FIG. 6C shows themass spectrum of myoglobin treated with a surfactant of the presentinvention, after degradation, exhibits a strong myoglobin signal.Without wishing to be bound by any particular theory, this result isbelieved to be due to at least two effects: 1) few, if any, micelles arepresent with the degraded surfactant of the present invention; and 2)fewer adducts of sample and the degraded surfactant of the invention areformed. These effects allow better sensitivity in mass spectrometry thanis possible when SDS is used.

After the chemical reaction, the molecules are released from thesurfactants of the present invention by degradation with acidicsolution. The molecules may be further purified by conventionalseparation methods such as liquid-liquid extraction, solid-phaseextraction or liquid chromatography. This ability to separate themolecules from surfactants easily after the chemical reaction may beused in various applications, with significant benefits to separationscience.

An additional embodiment of the invention provides a method forenhancing chemical digestion of a biomolecule comprising contacting themolecule with a digestive enzyme, e.g., a protease, and a surfactant ofthe present invention, to thereby enhance the chemical digestion of themolecule. In an additional embodiment, the invention provides a kit forenhancing chemical digestion of a biomolecule comprising a surfactant ofthe present invention, and instructions for use. In certain embodiments,the biomolecule is a protein. Exemplary proteins include, but are notlimited to, bovine serum albumin, lysozyme, ovalbumine, myoglobin,ubiqutin, and bacteriorhodopsin in certain embodiments, the kit forenhancing chemical digestion of a biomolecule further comprises adigestive enzyme, e.g., a protease. Exemplary proteases include, withoutlimitation, Trypsin, Chymotrypsin Lys-C, Glu-C (V8 protease), AspN,Arg-C, S. Aureus, Clostripain, Pepsin, and Papain.

In another embodiment, the invention provides a method of capturing alipophilic compound comprising contacting a lipophilic compound with asurfactant of the present invention, and degrading the surfactant toproduce hydrophobic compound, e.g., by contact with an acidic solution,to thereby capture the lipophilic compound. The lipophilic compound maybe contained in a sample, e.g., a biological sample. In certainembodiments, the product of degradation is compatible with massspectrometric detection, high performance liquid chromatographyanalysis, and with protease activity.

In addition, the lipophilic compound can be a protein fragment orpeptide. In certain embodiments, the protein fragment is generated bychemical digestion or a combination of chemical alteration and chemicaldigestion. In specific embodiments of the invention, the proteinfragment or the peptide is the product of a protein that has beendigested by contact with a protease and a surfactant of the presentinvention.

Another embodiment of the invention provides a method for enhancingsurface desorption ionization analysis, e.g., MALDI-MS, DIOS, or SELDI,of a molecule, e.g., a biomolecule, comprising solubilizing a moleculeby contacting the molecule with a surfactant of the present invention,to thereby analyze the sample by surface desorption ionization. Thesurfactant can be degraded prior to analysis, but the degradation is notrequired. Therefore, in certain embodiments of the invention, thesurfactant is degraded prior to analysis, while in other embodiments ofthe invention the surfactant is not degraded prior to analysis.

In addition, better recoveries are expected for hydrophobic proteins orproteins that are proteolysis resistant. Analysis of a complex mixturemay be difficult due to the large number of peptide fragments. Having away to reduce the number of fragments by fractionating a smallpercentage of them could provide some benefit. Additionally, once thesample is segregated into two distinct classes, the HPLC separation canbe optimized for each resulting class of peptides, thereby potentiallyresulting in additional information.

In another embodiment, SDS that has bound to the proteins duringSDS-PAGE can be replaced with the surfactants of the invention bywashing with buffers containing a surfactant of the invention, e.g.,ALS.

EXEMPLIFICATION Example 1 Preparation of sodium 4-[(2-methyl-2undecyl-1,3-dioxolan-4-yl)methyl]-1-propanesulfonate(3, 4) [ALS]

This example describes the preparation of certain anionic surfactants ofthe present invention. Various modifications to the following procedureswill be routine to one of ordinary skill in the art, in light of theteachings herein. For example, in the following procedures, toluene maybe substituted for benzene. In addition, any solvent that provides asufficient yield may be used in the recrystallization step.

1. Synthesis of 4 hydroxymethyl-2-methyl-2-undecyl-1,3-dioxolane (1, 2)

Firstly, 100 g (0.5 mol) of 2-tridecanone (Aldrich P/N 17, 283-9), 56 g(0.6 mol) of glycerol (Aldrich P/N 32,00-5), 200 mL of benzene, and 1.8grams of p-toluenesulfonic acid (Aldrich P/N 40,2885) were placed in a500 mL round bottom flask fitted with a Dean Stark apparatus. Themixture was heated to reflux with stirring until no further separationof water appeared. The reaction mixture was cooled to room temperatureand washed successively with a 100 mL portion of 5% sodium carbonatesolution and three 100 mL portions of water. The organic layer was driedover sodium sulfate, filtered and the benzene was removed with a rotaryevaporator. The residual oil was fractionated by distillation underreduced pressure to give the desired product (b.p. 140° C./0.3 mm Hg).The identity of the product was confirmed by ¹H NMR in CDCl₃.

2. Synthesis of ALS

50 g (0.18 mol) of 4-hydroxymethyl-2 methyl-2 undecyl-1,3-dioxolane, 8 g(0.2 mol) of powdered sodium hydroxide and 200 mL of benzene were placedin a 4 neck 500 mL flask fitted with a condenser, mechanical stirrer anda thermometer. The suspension was stirred at a constant 50° C. while 25g (0.2 mol) of 1,3-propanesultone (Aldrich P/N P5, 070-6) was slowlyadded over 30 minutes. The suspension was then stirred at 70-75° C. forat least 6 hours. Upon completion, the reaction mixture was poured into500 mL of boiling ethanol. The volume of the resulting mixture was thenreduced in vacuo with a rotary evaporator, producing a solid residuethat was subsequently dissolved in boiling ethanol and hot filtered.

The solid residue was additionally extracted with boiling ethanol, whichwas combined with the mother liquor. The solvent was removed in a rotaryevaporator, and the resulting residue was then recrystallized fromethanol to yield the product. Identity of the product was confirmed by¹H NMR in D₂O.

Example 2 Trypsin Activity Assay

Trypsin activity measurements were carried out by introducing 1 μg/ml oftrypsin to 0.25 mM N-α-benzoyl-L-arginine ethyl ester (BAEE, pH 7.9) atroom temperature. Changes in trypsin activity were plotted by measuringthe rate of hydrolysis of BAEE to a UV active product, BA, at A 252 nm.The analysis, shown in FIG. 1 and Table 1, indicates that SDS inhibitstrypsin activity at each the percentages examined. However, the additionof ALS to the trypsin solution containing SDS, shows an increase intrypsin activity, indicating a reactivation of trypsin digestion withinthe solution.

TABLE 1 Trypsin Activity Summary Trypsin solution* Trypsin activity(%)** No additive 100 0.1% ALS 99 0.5% ALS 87 0.1% SDS 20 0.5% SDS 10.1% SDS/0.1% ALS 58 50% Methanol 31 50% Acetonitrile 92 2M Urea 85 4MUrea 71 *0.5 μg of trypsin in 50 mM ammonium bicarbonate, pH 7.9; 0.2 mMof BEAA **Measured as delta BEAA absorbance @253 (slope within 5 min)

Example 3 Trypsin Digestion of Various Proteins

Proteins (2-20 μM), indicated in Table 2, were solubilized in 50 mMNH₄HCO₃ or 0.1-0.25% (w/w) ALS before tryptic digestion. The trypsin toprotein ratio used was 1-2%. The protein (˜17 μM) was solubilized with8M urea or 0.25% ALS. After tryptic digestion, ALS was destroyed rapidlyby addition of strong acid such as HCl or TFA. The by-products wereremoved by centrifugation prior to MS analysis. Protein digests weremass analyzed either by a MALDI.TOF mass spectrometer (M@LDI™ LR,Micromass UK Ltd) or separated by RP-HPLC (CapLC®, Waters Corporation)then interfaced to an orthogonal acceleration TOF mass spectrometer(LCT™, Micromass UK Ltd) via an electrospray ionization source.

FIG. 2 shows the LC/MS TIC of tryptic digested myoglobin solubilizedwith (A) 0.1% ALS or (8) 50 mM NH₄HCO₃ (no ALS). Myoglobin treated with0.1% ALS was observed to undergo complete tryptic digestion within 5minutes. Myoglobin in 50 mM NH₄HCO₃ was resistant to tryptic digestion.In fact, a majority of myoglobin in the sample without ALS remainedundigested after 9 hrs.

In addition, MALDI-TOF mass spectrometry was also performed on thedigested sample, prior to degradation of the surfactant (i.e., beforeaddition of acid). FIG. 3 shows the MALDI-TOF mass spectrometry analysisof the tryptic digestion of myoglobin after 5 minutes solubilized with0.1% ALS or 50 mM NH₄HCO₃ (no ALS).

TABLE 2 Protein Digestion by Trypsin Digestion Protein Difficulty w/oALS With ALS (0.1%) BSA (66K) Easy minutes 16 peptides + minutes 21peptide

intact protein Lysozyme (16K) Easy minutes 2 peptides + minutes  6peptides intact protein Ovalbumine (43K) Moderate minutes 0 peptides +minutes  2 peptides intact protein Myogiobin (17K) Moderate minutes 0peptide + minutes 12 peptides intact protein Ubiqutin (8K) Difficultminutes 0 peptide + minutes  9 peptides intact protein BacteriorhodopsinVery Overnight 1 peptide (weak Overnight  3 peptides (26K) Difficultsignal) + intact protein Proteins are not reduced MALDI-TOF (900-3000amu)

indicates data missing or illegible when filed

Direct Analysis of Sample, No Work Up (No ZipTip™)

Furthermore, Table 2 indicates not only was the rate digestion enhanced,but also, more complete, in that the number of peptide fragmentsgenerated was significantly increased.

Example 4 Comparative Digestion of Myoglobin with Various Proteases

Myoglobin comparative digestion was performed as in Example 3 (in thepresence and absence of ALS), with the exchange of the followingproteases for trypsin:

A. Asp-N (FIG. 4A) B. Lys-C (FIG. 4B) C. Glu-C (FIG. 4C)

Digestions were performed for about 50 minutes. The enzymes werepurchased from Roche Diagnostics. Analysis is shown in FIGS. 4A-4C, asindicated above. In each case, when no ALS was in the digestionsolution, no observable digestion took place. However, with ALS, withinabout 50 minutes, the entire protein was consumed.

Example 5 In Solution Protein Digestion Using Immobilized Trypsin

Trypsin immobilized on agarose beads may be purchased from Pierce. Thebeads (100 μl, 20 units of enzyme activity) is pipetted into a vial andis washed with 50 mM ammonium acetate. The excess buffer is thenremoved, followed by the addition of 100 μg of Myoglobin dissolved in100 μl of 50 mM ammonium acetate buffer, pH 7.9. The reaction mixture ismixed vigorously for 5 minutes, the vial is briefly centrifuged toseparate beads from solution, and the clear liquid is then used foranalysis. MS and HPLC are used to monitor the content of myoglobin andgenerated peptides.

This experiment is then repeated with an addition of ALS to the reactionsolution. Final concentration of ALS was 0.1% (w/v). The rate ofenzymatic reaction is compared with the experiment performed in theabsence of ALS.

Example 6 On-Line Protein Digestion Using Trypsin Reactor

Trypsin immobilized on agarose beads may be purchased from Pierce.Flow-through devices may be constructed in order to demonstrate on-lineprotein digestion.

A gel loading tip is crimped at the bottom and is filled with slimy ofagarose beads with immobilized trypsin. The excess liquid (50% glycerol,0.05% sodium azide) can then removed while the beads are entrapped inthe tip. The length of the bead is ˜2 centimeters.

The tip is then filled from the top with an excess of 50 mM ammoniumacetate buffer and is washed several times using moderate positivepressure generated by pipette or by centrifugation. Myoglobin (100 μg)is dissolved in 100 of 50 mM ammonium acetate buffer, pH ˜7.9 is placedon top of the beads layer and slowly is passed through the bead usingeither positive pressure or low speed centrifugation. The experiment iscomplete within 30 seconds. The solution is collected into a reservoirand analyzed off-line by MS and HPLC.

On-Line Setup

This experiment is then repeated using similar setup with 100 μg ofMyoglobin dissolved in 100 μl of 50 mM ammonium acetate buffercontaining 0.1% ALS (w/v). The speed of digestion is compared to thecontrol ALS free experiment.

The on-line setup is constructed using cartridge housing (e.g. 20×2.1mm) packed with agarose beads with immobilized trypsin. The proteinsample is then injected and is washed from the cartridge with 50 mMammonium acetate buffer directly onto the analytical column packed withRP-HPLC sorbent. The trapped peptides are eluted from the main columnwhile trypsin reactor is being washed and equilibrated with freshammonium acetate buffer.

Example 7 Mass Spectrometric Detection of Myoglobin Treated with ALS orSDS

In this example, mass spectroscopy is used to compare a surfactant ofthe present invention to SDS.

Mass spectrometric detection was performed on myoglobin using a PlatformLC (Micromass, Manchester, UK) with constant infusion. A stock solutionof 50 μM horse skeletal muscle myoglobin (Sigma. P/N M0630) was preparedin 50/50 20 mM ammonium acetate, pH 5.1/acetonitrile (v/v). A stocksolution of 1% SDS and 1% ALS were also prepared in both 50/50 ammoniumacetate/acetonitrile, and 40/40/10 ammonium acetate/acetonitrile/glacialacetic acid. The surfactant stock solutions were both prepared fresh,and allowed to sit for at least 16 hours. To 100 μL of each of thesurfactant stock solutions was added 100 μL of myoglobin stock and 800μL 50/50 ammonium acetate/acetonitrile. Each solution was then drawninto a 1 mL syringe and placed in a syringe pump (Harvard Instruments).The solution was infused into the mass spectrometer at a flow rate of 20μL/min. Mass spectrometer settings were as follows:

Gas flow rate: 340 L/h; Source temperature: 100° C., Capillary voltage:3.46 kV; Cone voltage: 25 V; Ion energy: 0.6; Scan rate: 3.0 sec/scan;Scan range: 250-1600 amu. Mass spectra are shown in FIG. 6. For the casewhere SDS is the surfactant, no signals are observed which are due tomyoglobin. The spectrum of the sample containing degraded ALS, on theother hand, exhibits a strong myoglobin signal.

Example 8 Mass Spectrometric of Disulfide Bond Reduction of Lysozymewith ALS

The disulfide bond of lysozyme C was reduced in the presence and absenceof 0.25% ALS. The mass spectrometry analysis of the disulfide bondreduction is shown in FIG. 5.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. A method for enhancing a chemical reaction of a molecule comprisingcontacting the molecule with a surfactant represented by formula I:

in which p is 0, for 2; R is alkyl; R₁ and R₂ are each, independently,hydrogen or methyl; and R₃ is selected from —OSO₃ ⁻, —R₄OSO₃ ⁻,—R₄OR₅SO₃ ⁻, and —OR₅SO₃ ⁻, wherein R₄ and R₅ are each, independently,lower alkyl; to thereby enhance the chemical reaction of the molecule.2. The method of claim 1, wherein the molecule is a biomolecule.
 3. Themethod of claim 2, further comprising analyzing the biomoleculefollowing the chemical reaction.
 4. The method of claim 2, wherein thebiomolecule is contained in a biological sample.
 5. The method of claim4, wherein the biological sample is selected from the group consistingof inclusion bodies, biological fluids, biological tissues, biologicalmatrices, embedded tissue samples, and cell culture supernatants.
 6. Themethod of claim 2, wherein the biomolecule is selected from the groupconsisting of a protein and a peptide.
 7. The method of claim 6, whereinthe biomolecule is selected from the group consisting of a lipophilicprotein, a receptor, a proteolytic protein, and a membrane-boundprotein.
 8. The method of claim 3, wherein the analysis is selected fromthe group consisting of solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry, liquid chromatography,liquid-liquid extraction, membrane extraction, soxhlet extraction,precipitation, clarification, electrochemical detection, staining,elemental analysis, Edmund degradation, nuclear magnetic resonance,infrared analysis, flow injection analysis, capillaryelectrochromatography, ultraviolet detection, and combinations thereof.9. The method of claim 8, wherein the mass spectrometry is surfacedesorption ionization mass spectrometry.
 10. The method of claim 9,wherein the surfactant is not degraded prior to analysis.
 11. The methodof claim 9, wherein the surfactant is degraded prior to analysis. 12.The method of claim 1, wherein the reaction comprises chemicaldigestion, chemical alteration, or a combination thereof.
 13. The methodof claim 12, wherein the reaction comprises chemical digestion.
 14. Themethod of claim 13, further comprising contacting the biomolecule with aprotease, CNBr, or hydroxylamine.
 15. The method of claim 13, furthercomprising separating the resulting biomolecule fragments.
 16. Themethod of claim 14, wherein the protease is immobilized.
 17. The methodof claim 14, wherein the protease is selected from the group consistingTrypsin, Chymotrypsin Lys-C, Glu-C (V8 protease), AspN, Arg-C, S.Aureus, Clostripain, Pepsin, and Papain.
 18. The method of claim 9,wherein the reaction comprises chemical alteration.
 19. The method ofclaim 18, wherein the chemical alteration is selected from the groupconsisting of alkylation, reduction, and a combination thereof.
 20. Themethod of claim 1, wherein the enhancement of the chemical reactioncomprises a favorable chemical property.
 21. The method of claim 20,wherein the favorable chemical property is selected from the groupconsisting of a more complete reaction, increased efficiency, increasedyield, increased rate, and increased utility.
 22. The method of claim12, wherein the chemical reaction comprises denaturing the biomolecule,solubilizing the biomolecule, or a combination thereof.
 23. The methodof claim 1, further comprising degrading the surfactant after thechemical reaction.
 24. The method of claim 23, wherein the surfactant isdegraded by contact with an acidic solution.
 25. The method of claim 1,wherein the surfactant is represented by formula II:

in which R₆ is alkyl; R₇ is selected from —OSO₃ ⁻, —R₄OSO₃ ⁻, —R₄OR₅SO₃,and —OR₅SO₃ ⁻, wherein R₄ and R₅ are each, independently, lower alkyl.26. The method of claim 1 wherein the surfactant has the followingchemical structure:


27. The method of claim 1 wherein the surfactant has the followingchemical structure:


28. The method of claim 1 wherein the enhancement of the chemicalreaction facilitates on-line automation, separation, mass spectrometricanalysis, or a combination thereof.
 29. The method of claim 1 whereinthe chemical reaction is performed under micro scale conditions.
 30. Themethod of claim 13 wherein the digestion occurs in an electrophoreticgel.
 31. The method of claim 30 wherein the digestion occurs in thepresence one or more surfactants that are different from the surfactantin Formula I.
 32. The method of claim 31 wherein the digestion occurs inthe presence of SDS.
 33. The method of claim 31 wherein the digestionoccurs in the absence of SDS.
 34. A method for analysis of a biomoleculecomprising: enhancing a chemical reaction of the biomolecule bycontacting a sample containing the biomolecule with a surfactantrepresented by the formula:

in which p is 0, 1 or 2; R is alkyl; R₁ and R₂ are each, independently,hydrogen or methyl; and R₃ is selected from —OSO₃, —R₄OSO₃ ⁻, —R₄OR₅SO₃⁻, and —OR₅SO₃ ⁻, wherein R₄ and R₅ are each, independently, loweralkyl; and analyzing the sample; to thereby analyze the biomolecule. 35.The method of claim 34, wherein the molecule is a biomolecule.
 36. Themethod of claim 35, further comprising analyzing the biomoleculefollowing the chemical reaction.
 37. The method of claim 35, wherein thebiomolecule is contained in a biological sample.
 38. The method of claim37, wherein the biological sample is selected from the group consistingof inclusion bodies, biological fluids, biological tissues, biologicalmatrices, embedded tissue samples, and cell culture supernatants. 39.The method of claim 35, wherein the biomolecule is selected from thegroup consisting of a protein and a peptide.
 40. The method of claim 39,wherein the biomolecule is selected from the group consisting of alipophilic protein, a receptor, a proteolytic protein, and amembrane-bound protein.
 41. The method of claim 36, wherein the analysisis selected from the group consisting of solid phase extraction, solidphase micro extraction, electrophoresis, mass spectrometry, liquidchromatography, liquid-liquid extraction, membrane extraction, soxhletextraction, precipitation, clarification, electrochemical detection,staining, elemental analysis, Edmund degradation, nuclear magneticresonance, infrared analysis, flow injection analysis, capillaryelectrochromatography, ultraviolet detection, and combinations thereof.42. The method of claim 41, wherein the mass spectrometry is surfacedesorption ionization mass spectrometry.
 43. The method of claim 42,wherein the surfactant is not degraded prior to analysis.
 44. The methodof claim 34, wherein the reaction comprises chemical digestion, chemicalalteration, or a combination thereof.
 45. The method of claim 44,wherein the reaction comprises chemical digestion. 46-61. (canceled) 62.The method of claim 45 wherein the digestion occurs in anelectrophoretic gel.
 63. The method of claim 62 wherein the digestionoccurs in the presence of SDS.
 64. The method of claim 62 wherein thedigestion occurs in the absence of SDS. 65-112. (canceled)
 113. A methodfor enhancing chemical digestion of a biomolecule comprising: contactingthe molecule with a digestive enzyme and a surfactant represented byformula I:

in which p is 0, for 2; R is alkyl; R₁ and R₂ are each, independently,hydrogen or methyl; and R₃ is selected from —OSO₃ ⁻, —R₄OSO₃ ⁻,—R₄OR₅SO₃ ⁻, and —OR₅SO₃ ^(−wherein R) ₄ and R₅ are each, independently,lower alkyl; to thereby enhance the chemical digestion of the molecule.114-122. (canceled)